Recombination cloning methods, such as Gateway™ (Life Technologies, Inc., Paisley, UK) are typically used to clone only one DNA molecule into an entry vector. In certain cases, it may be desirable to create a chimeric or modified DNA molecule, consisting of two parts, for cloning in a Gateway™ entry vector. Examples of such chimeric molecules include suitable gene-promoter combinations, genes encoding a fusion protein, etc. Examples of modified DNA molecules include for example site directed mutants. Classical cloning techniques comprise the making of the chimeric molecule, for example a promoter-gene combination, in a separate intermediate construct, followed by the cloning of the chimeric molecule as a whole into the entry vector.
The Gateway™ cloning system was designed to introduce the DNA of interest via site specific recombination into an “entry clone”, whereby the DNA molecule is cloned as such that it is easily introduced via another site specific recombination reaction into a subsequent set of “destination vectors”. These latter vectors carry the necessary backbone sequences to successfully transform a particular host cell and to assure the desired expression of the cloned DNA of interest. Before the DNA molecule, for example the promoter-gene combination is introduced in the “entry clone”, the gene must be operably linked to the promoter and the correct promoter-gene combination must be selected. Classical cloning steps to achieve this operably linkage, typically involve (i) cleavage of fragments via restriction enzymes or amplification of overlapping fragments via PCR techniques and (ii) subsequent assembling of the different fragments into an intermediary construct via ligation of the restriction enzyme sites or via assembly or “fill-in” PCR, (iii) analyzing the intermediary constructs and (iv) selecting the correct promoter-gene combination. From this intermediary construct the desired DNA molecule, here the promoter-gene combination will be PCR amplified as 1 whole, using two PCR primers that contain the suitable Gateway™ recombination sites. These recombination sites are compatible with the recombination sites of the acceptor plasmid (the entry clone or a destination clone of the Gateway™ system) and the amplified and purified DNA molecule as a whole will be recombined into the acceptor plasmid. One way to simplify this method would be to make the combination of the promoter-gene molecule simultaneously with the recombination cloning into the Gateway™ plasmid.